The long term objectives of this project are to understand the function and regulation of protein phosphorylation in the control of cell growth and differentiation, and the molecular mechanisms of growth factor- induced signal transduction. The Ras/mitogen activated protein kinase (MAPK, also known as ERK for extracellular signal regulated kinase) pathway plays an essential role in mediating the signal transduction of mitogenic growth factors which act through tyrosine kinase receptors. The GTP-bound form of Ras is active while the GDP-bound form is generally believed to be inactive. Activation of Ras leads to activation of the ERK cascade which relays the signal from tyrosine kinase receptors to activation of nuclear transcription factors. Components directly involved in the Ras/ERK pathway have been identified, however, this pathway is subject to multiple levels of regulation. Preliminary data demonstrate that expression of a dominant negative Ras mutant, a GDP-bound form of Ras, specifically inhibits the phosphorylation and activation of Elk-1, a physiological substrate of ERK. This inhibition is apparently not through a direct inhibition of ERK. Additional data indicated that the inhibitory effect of dominant negative Ras may be mediated by KSR, a kinase identified by genetic screens as a mediator of Ras function. The specific aims of this proposal are: 1). to determine the molecular mechanism of a novel function of Ras in regulation of Elk-1 activation and to demonstrate whether this function of Ras depends on the GDP form of Ras; 2). To elucidate the biochemical mechanisms of KSR in signal transduction of the MAP kinase pathway and to establish the relationship of KSR and Ras in Elk-1 regulation; 3). To understand the molecular basis for the specificity of MEK in activation by upstream activators and recognition of downstream substrates. Biochemical, and cell and molecular biological approaches will be used to achieve these goals. Completion of this proposal will further our understanding of the Ras/MAP kinase signal transduction.